WO2020089934A1 - Procédé biocatalytique pour la racémisation de d-éphédrine - Google Patents

Procédé biocatalytique pour la racémisation de d-éphédrine Download PDF

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Publication number
WO2020089934A1
WO2020089934A1 PCT/IN2019/050797 IN2019050797W WO2020089934A1 WO 2020089934 A1 WO2020089934 A1 WO 2020089934A1 IN 2019050797 W IN2019050797 W IN 2019050797W WO 2020089934 A1 WO2020089934 A1 WO 2020089934A1
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ephedrine
rotatory
racemization
dextro
reaction
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Ganapati Dadasaheb Yadav
Shalaka Sanjiv MOHIRE
Rajendran ASHILY
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C209/00—Preparation of compounds containing amino groups bound to a carbon skeleton
    • C07C209/68—Preparation of compounds containing amino groups bound to a carbon skeleton from amines, by reactions not involving amino groups, e.g. reduction of unsaturated amines, aromatisation, or substitution of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)

Definitions

  • the present invention relates to a process for racemization of dextro-rotatory ephedrine via biocatalytic transformation and is particularly useful for conversion of dextro- rotatory ephedrine (d- ephedrine) to racemic mixture of dextro-rotatory ephedrine (d-ephedrine) and levo-rotatory ephedrine (l-ephedrine).
  • the present invention provides an effective process to recover the levo-rotatory ephedrine that exhibit potential bronchodilatory and anti -hypotensive activities.
  • racemic ephedrine results into formation of detro- rotatory ephedrine (+ephedrine) along with the levo-rotatory ephedrine (- ephedrine), of which the latter one, l-form is comparatively a more active compound.
  • the d-form cannot be utilized in pharmaceutical companies and thus remains as an undesirable compound.
  • SCHEME 1 Structure of (1) levo-rotatory ephedrine, (2) dextro-rotatory ephedrine.
  • the IUPAC name of the enantiomeric compounds 1 and 2 are (lR,2S)-2- methylamino- 1 -phenylpropan- 1 -ol and ( 1 S,2R)-2-methylamino- 1 -phenylpropan- l-ol, respectively.
  • Racemization is the process when the enantiomer is converted into racemic modification (i.e. chemical reactions) or when one pure form of an enantiomer is converted into equal proportion of both enantiomers (lcvo- and dcxtro-). forming a racemate.
  • the second strategy is asymmetric synthesis: the use of various techniques to prepare the desired compound in high enantiomeric excess. Techniques encompassed include the use of chiral starting materials (chiral pool synthesis) the use of chiral auxiliaries and chiral catalysts, and the application of asymmetric induction.
  • racemic ephedrine is mixture of dextro-rotatory ephedrine (+ephedrine) along with the levo-rotatory ephedrine (-ephedrine), of which the latter one, levo-form is comparatively a more active compound.
  • the kinetic resolution techniques like crystallization and enzymatic resolution are well established techniques for chiral separation of desired enantiomer from racemic mixture. After such resolution, the dextro-form cannot be utilized in pharmaceutical companies and thus remains as an undesirable compound.
  • existing methods mainly comprise of chemical processes which followed the harsh reaction conditions, while few of them were consistent with mild conditions, usually using the transition metal catalysts.
  • Racemization of enantiomeric ephedrine was carried on by treating it with higher temperature above the normal with an alkali alcoholate.
  • l-aryl-aklylamines were subjected to racemization by using metal alkoxides in dimethyl sulphoxides optionally in the presence of appropriate alcohols.
  • the known racemization processes are not suitable for industrial scale due to unsatisfactory yield or insufficient quality of products or industrially disadvantageous catalysts or some working up problems.
  • this approach is made towards biocatalysis with lower temperature range (0-110 °C), lower energy consumption, lesser formation of by-products, non-toxic and safer process when used correctly, reusable, biodegradable, stereo- and regio- selective unlimited production capabilities, industrially scalable, economic process and having good yield in terms of racemic compound which is on demand.
  • the inventors of the present invention developed an improved method for converting a dextro-rotatory ephedrine to racemate of ephedrine (Both levo and dextro form in mixture), further separating levo- rotatory ephedrine from the reaction mass as active compound and reusing the separated out dextro-rotatory ephedrine for same racemization reaction process.
  • the present invention provides a biocatalytic process for racemization of dextro-rotatory ephedrine, wherein whole cell enzyme biocatalysis has been used which has an add-on advantage in the racemization process. By using the whole cells, the cost of process of extraction, purification, separation and maintenance of specific enzymes can be minimized.
  • the present invention is a solution for converting the undesired d-ephedrine produced during synthesis of ephedrine into a racemic mixture to again recover the desirable l-form of ephedrine from it.
  • the d-ephedrine is made into a reusable form.
  • the main objective of present invention is to provide a process for racemization of dextro-rotatory ephedrine via biocatalytic transformation.
  • the present invention relates to a process for racemization of dextro-rotatory ephedrine via biocatalytic transformation and is particularly useful for conversion of dextro- rotatory ephedrine (d- ephedrine) to racemic mixture of dextro-rotatory ephedrine (d-ephedrine) and levo-rotatory ephedrine (l-ephedrine).
  • the present invention provides a process for Racemization of dextro- rotatory ephedrine, the process comprising of:
  • Step 1 Preparation of suspension of Rhizopus Oryzae fungi pellets in diammonium phosphate buffer having pH in the range of pH 5 to 9
  • Step 2 Ultrasonicating the step 1 suspension for time between 3 to 10 min
  • Step 3 Addition of d-ephedrine in ultrasonicated suspension of step 2) and carrying out reaction at temperature between 25 to 35 °C for 10 to 36 h under constant stirring,
  • Step 4 Separation of liquid from solid mass by centrifugation at 300 to 10000 rpm.
  • concentration of Di-ammonium phosphate is between 5 to 55mM and d- ephedrine concentration is in range between 5 to 500mM.
  • the sonication frequency is between 10 Hz to 100 Hz and specifically inoculum to substrate ratios (ISRs) used for reaction is between 0.1 to 3.
  • ISRs inoculum to substrate ratios
  • This invention relates to process for racemization of dextro-rotatory ephedrine via enzymatic kinetic conversion, and is particularly useful for conversion of dextro rotatory ephedrine (d- ephedrine) to racemic mixture of dextro-rotatory ephedrine (d-ephedrine) and levo-rotatory ephedrine (l-ephedrine) that exhibit potential bronchodilatory and anti -hypotensive activities.
  • dextro rotatory ephedrine dextro rotatory ephedrine
  • l-ephedrine levo-rotatory ephedrine
  • racemic ephedrine results into formation of detro- rotatory ephedrine (d-ephedrine) along with the levorotatory ephedrine (1- ephedrine), of which the latter one, l-form is comparatively a more valuable compound.
  • the d-form cannot be utilized in pharmaceutical companies and thus remains as an undesirable compound.
  • racemizing the d-ephedrine and further subjecting it to any of the known technique for resolution of racemic mixture it is possible to recover l-ephedrine again from it.
  • the present invention is a greener approach for utilizing the undesirable d- ephedrine produced during synthesis into a desirable l-ephedrine by developing a simple process using biocatalysis phenomena.
  • the process is developed to convert d-ephedrine into racemic mixture of d- and l-ephedrine.
  • This racemization required the inversion of functional groups present on the chiral carbon atom of d-ephedrine molecule.
  • the overall reaction is described in scheme 2 as follows;
  • the treated whole cells of the organisms were incubated with the substrate for certain period of time which resulted into total racemization of d-ephedrine (mixture of 50% d- ephedrine and 50% l-ephedrine).
  • the said invention of biocatalytic process for racemization of d-ephedrine is very simple, environmental friendly and highly economic process which can be easily scaled up for industrial use.
  • the suitable culture media helps in expressing the particular enzymes in whole cells for racemization of d-ephedrine. Pure racemic d and l-ephedrine is obtained without formation of other undesired by-products like pseudo-ephedrine. The total amount of d-ephedrine introduced in bio-reactor is totally racemized into d, l-ephedrine respectively. Hence the problem of batch variation is not significant even though using the treated whole cell enzymes instead of specific extracted and purified enzymes. Thus, it can prove to be an easily scalable, cost-effective and a green process for racemization of d-ephedrine.
  • the f ingal strain was subcultured, grown, and the enzymes that are responsible for racemization were expressed in suitable media which can be grouped into three parts: Part I: Potato Dextrose Agar (PDA) Media, potato dextrose 0.05 to 1 g, Agar 0.05 to 1 g and de-ionized water. Part II: Solid media: 0.4 to l0 %glucose or sucrose or galactose and other such nutrients can be used, 1 to 5 % yeast extract or malt extract, 0.05 to 1 % peptone or other substrates can be used, of which peptone gave good results and 1 to 10 % agar.
  • PDA Potato Dextrose Agar
  • Solid media 0.4 to l0 %glucose or sucrose or galactose and other such nutrients can be used, 1 to 5 % yeast extract or malt extract, 0.05 to 1 % peptone or other substrates can be used, of which peptone gave good results and 1 to 10 % agar.
  • Liquid media 0.5 to 10 % glucose or galactose or starch or sucrose and other similar nutrient compounds can be used, 0.05 to 2 % yeast extract or beef extract or malt extract can be taken, 0.01 to 2 % sodium chloride or potassium chloride or similar salts can be used and about 0.01 to 5 % tryptone or casein peptone can be taken.
  • Strains of R. Oryzae were first subcultured and grown on PDA slant from 5 h up to 5 days at around 20 to 40 °C on petri plate in an aseptic area, followed by preparation of solid media. The media components were autoclaved and poured into the petri plates.
  • Part I The cells were treated with ultrasonication and the broth was kept as a control for incubation; Part II: The cells were untreated with ultrasonication, the substrate d-ephedrine was added and kept as another control for incubation;
  • Part III The cells were treated with ultrasonication, the substrate d-ephedrine was added and kept as reaction mass for incubation;
  • the reaction was carried out for different concentrations of substrate d- ephedrine and was studied for racemization.
  • the total racemization of d- ephedrine was obtained which gave up to 50-50 ratio of d- ephedrine and 1- ephedrine, respectively.
  • Example 1 Racemization using Chemical catalysis- Racemization of d- ephedrine using modified heterogeneous nanocatalysts, external hydrogen pressure and different solvents:
  • Detection Method The chiral HPLC method was developed for the separation of the d and 1 enantiomer from the racemic mixture of the dl-ephedrine as well as for the d-ephedrine standard in water.
  • the catalyst Pd/AlO(OH) shows conversion of d-ephedrine to d,l- ephedrine to some extent in water as solvent. No product was formed on increasing the temperature of the reaction at 4 h. Role of solvent in the system is also important.
  • Example 2 Enzyme catalysis- Whole cell biocatalytic racemization of d- ephedrine to 1-ephedrine using Rhizopus Oryzae.
  • Stepl The cultures of Rhizopus species, Aspergillus species and Escherichia coli were selected, subcultured and stored in refrigerator at 4 °C which can be used as an enzymatic whole cell biocatalyst for bioconversion of racemic ephedrine.
  • the fungal and bacterial cultures were maintained on suitable growth media.
  • Rhizopus Oryzae which can be used in enantioselective production were grown in solid medium containing 2% glucose, 2% yeast extract, 0.1% Peptone and 2% Agar
  • the plates were incubated at 30°C for 3-4 days for spore production.
  • reaction was carried out on an orbital shaker at 30°C for 24 hrs.
  • E. 50 mg d-ephedrine was added in 10 mL of buffer solution along with ultrasonicated whole cells and kept for incubation at 20- 60 °C, from 0 to 200 rpm for 6 h to 5 days. Samples were collected at every 6 h reaction time. It was observed that the reaction samples gave total racemic mixture of d- and 1- ephedrine (50:50) at 12 h.
  • example 3 (A) to (G) the concentration of the substrate is varied while keeping the buffer volume same.
  • example 3(G) we tried to increase the volume of buffer along with the substrate concentration.
  • the dextro- rotatory ephedrine gets racemized into detro- and levo- rotatory ephedrine in 12 h.
  • Example 4 Effect of sonication and Surfactant: A control experiment was performed using whole cell microorganisms with the pre-treatment.
  • Ultrasonic action in a whole cell suspension causes cell disruption and conformational changes in protein structure of enzymes. These conformational structure may cause increase in enantioselectivity of ephedrine.
  • Tween 80 is also used for disruption and dispersion of fungal cultures for release of enzymes but it also mix up with the substrate and creates problem in separation and purification of desired compound.
  • DAP Diammonium phosphate
  • Table 3 Interpretation of HPLC % area conversion data for d-ephedrine racemization where,‘+’: d-ephedrine, 1-ephedrine.
  • Racemization of d-ephedrine was achieved successfully in buffer using pretreated whole cells of R. oryzae. While Aspergillus oryzae, R. oligosporous, R. stolonifer had no effect on d-ephedrine even after 24 h of reaction time.
  • Rhizopus species Strains give racemization of d-ephedrine.
  • Example 6 Effect of Enzyme Activity in reaction mass: The fungal strain consists of a large number of enzymes of which only few enzymes are active and take part in the reaction. Lipase enzyme and Alcohol Dehydrogenase (ADH) generally playing role in racemization of d-ephedrine. This study was carried to identify the concentration of enzymes required particularly for racemization of d- ephedrine. A. Lipase Activity
  • Phenolphthalein indicator was added and the mixture was titrated against the standard 0.1 N NaOH solution.
  • ADH Alcohol dehydrogenase
  • ADH activity was analyzed spectroscopically by measuring the increase in absorbance at 340 nm promoted by formation of NADH during oxidation of ethanol. • Only liquid part of homogenized cell suspension was used in ADH activity. These cell suspensions were filtered with appropriate filter.
  • ADH activity was measured in 3 mL cuvette containing 50 pL of 20 mM ethanol, 1 mL of distilled water and 1.25 mL of 13 mM b-NAD.
  • the spectrophotometric absorbance of cell suspension was carried out at specific interval time for about 12 h reaction.
  • Mobile phase 940 ml of buffer and 60 ml of methanol, filter through 0.45 m filter and degas.
  • Buffer Weigh accurately 11.6 g of ammonium acetate and dissolve and dilute to

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Abstract

La présente invention concerne des procédés de racémisation d'éphédrine dextrogyre par conversion cinétique enzymatique, qui sont particulièrement utiles pour la conversion d'éphédrine dextrogyre (d-éphédrine) en un mélange racémique d'éphédrine dextrogyre (d-éphédrine) et d'éphédrine lévogyre (l-éphédrine) pour récupérer l'éphédrine lévogyre qui présentent des activités potentielles bronchodilatatoires et anti-hypotension. Le procédé permet d'obtenir une suspension de pastilles de champignons Rhizopus Oryzae dans une solution tampon de phosphate de diammonium ayant un pH dans la plage de 5 à 9 et une sonication efficace pour extraire des enzymes spécifiques pour une inversion de groupes fonctionnels présents sur l'atome de carbone chiral de la molécule de d-éphédrine à basse température qui présente des avantages d'activité à basse température (20 à 50 °C), une faible consommation d'énergie, et moins de formation de sous-produits.
PCT/IN2019/050797 2018-11-02 2019-10-30 Procédé biocatalytique pour la racémisation de d-éphédrine Ceased WO2020089934A1 (fr)

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IN201821041686 2018-11-02
IN201821041686 2018-11-02

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2214034A (en) * 1939-09-05 1940-09-10 Abbott Lab Racemization of optically active compounds
WO1989009765A1 (fr) * 1988-04-07 1989-10-19 Sepracor, Inc. Systemes de resolution enzymatique et composes utilises dans ces systemes, et leurs preparations
WO2007090767A1 (fr) * 2006-02-09 2007-08-16 Basf Se Racémisation biocatalytique d'alpha-hydroxycétones

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2214034A (en) * 1939-09-05 1940-09-10 Abbott Lab Racemization of optically active compounds
WO1989009765A1 (fr) * 1988-04-07 1989-10-19 Sepracor, Inc. Systemes de resolution enzymatique et composes utilises dans ces systemes, et leurs preparations
WO2007090767A1 (fr) * 2006-02-09 2007-08-16 Basf Se Racémisation biocatalytique d'alpha-hydroxycétones

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